Defining mechanisms limiting NK cell function in the multiple myeloma tumor microenvironment

Natural killer (NK) cells are cytotoxic cells that have an intrinsic ability to mediate anti-tumor responses. However, the immunosuppressive tumor microenvironment (TME) can limit their efficacy. Multiple myeloma (MM) is a hematologic malignancy characterized by clonal proliferation of plasma cells within the bone marrow. There is a growing understanding that the tumor microenvironment (TME) is a critical factor influencing the effectiveness of cellular therapies. A hallmark characteristic of rapidly proliferating cancer cells is the production of a high amount of lactic acid. Myeloma cells also produce metabolites, including lactic acid, that suppress the effector function of T cells. How lactic acid affects NK cell function remains poorly understood. In this study, we hypothesize that increased lactic acid in the TME inhibits NK cell functionality and thereby serves as a key TME checkpoint on NK cell anti-tumor responses. To test this, primary conventional (c)NK cells and ML NK cells were exposed to varying concentrations of lactic acid and assessed for cytotoxicity, proliferation, and cytokine production in vitro. We observed that cNK cell IFNᵧ production (P=0.0035) significantly decreased with 15mM lactic acid conditions compared to controls (0mM and 3mM). ML-differentiation resulted in a partial rescue of lactic acid-induced reductions in IFNᵧ production (p=0.0417) observed in cNK cells. Next, we found that degranulation (measured via sCD107a) was significantly (P=0.0018) reduced in both cNK and ML NK cells under 15mM lactic acid treatment conditions compared to controls (0mM and 3mM). cNK and ML NK cytotoxicity were significantly decreased in 15 mM lactic acid compared to controls, with ML NK cells again exhibiting partial resistance to lactic-acid compromised killing. To investigate the impact of lactic acid on NK cell proliferation, we activated the NK cells with IL-12, IL-15, and IL-18 overnight and labeled them with cell trace violet (CTV) . CTV-labeled cells were incubated in media with lactate conc. By day 7, NK cell proliferation was abrogated with exposure to 15 mM lactic acid, while 40% of NK cells proliferated in control conditions (0mM or 3mM lactic acid). This data suggests that NK cell proliferation are suppressed with high lactic concentration. Our data further revealed that lactic acid (15mM) markedly suppressed IL-15-induced pSTAT5 signaling in cNK cells. In contrast, ML NK cells maintained higher pSTAT5 levels in response to IL-15 under high lactic acid (15mM) conditions, demonstrating greater resilience to this TME stress. Finally, we identified the acidity as the major driver of NK cell dysfunction induced by lactic acid by comparing it to sodium lactate and matched pH control conditions. These data suggest that lactic acid is a metabolic checkpoint on NK cells, and ML differentiation and strategies to insulate NK cells from lactic acid effects may improve NK cell anti-tumor responses.